1. Field of the Invention
The present invention relates to novel fluorescent-labeled or chemiluminescent-labeled conjugates for use in specific binding assays for ligands (e.g., antigens and haptens) or receptors (e.g., antibodies, specific binding proteins, and cell surface receptors). The invention further relates to intermediate compounds produced in the synthesis of the novel labeled conjugates, and kits containing such labeled conjugates and/or intermediates.
2. Discussion of the Background
Presently, several immunochemical methods exist for the detection of ligands such as haptens, antigens or antibodies. Radioimmunoassay is a widely used method. This method employs a radioisotope-labeled antigen (ligand) to compete with the antigen from a test sample for binding to a specific antibody. U.S. Pat. No. 3,709,868 describes such a radioimmunoassay. While by definition, radioimmunoassay is based on the binding of a specific antibody with an antigen or hapten, radioactive binding assays which are based on other specific binding interactions, such as between hormones and their binding proteins, have also been developed.
Several non-isotopic immunoassays have been proposed to eliminate the disadvantages associated with radioactive materials. Ligands such as an antigen or hapten and receptors such as an antibody have been labeled with a variety of non-radioactive compounds, including chemiluminescent and fluorescent molecules.
Specific examples of useful chemiluminescent labels are disclosed in German OLS No. 2,618,511 and include luminol (3-aminophthalhydrazide or 5-amino-2,3-dihydro-1,4-phthalazinedione) and isoluminol (4-aminophthalhydrazide or 6-amino-2,3-dihydro-1,4-phthalazinedione). The use of N-(4-aminobutyl)-N-ethylisoluminol (6-[N-(4-aminobutyl)-N-ethylamino]-2,3-dihydrophthalazine-1,4-dione) as a chemiluminescent label is reported in Simpson et al., Nature, Vol. 279, p. 646 (1979). The preparation of chemiluminescent phthalhydrazide labeled ligands is described in U.S. patent application Ser. No. 927,621, filed July 24, 1978, entitled "Chemiluminescent Phthalhydrazide Labelled Conjugates." U.S. Pat. No. 4,331,808 discloses labeling ligands with chemiluminescent naphthalene-1,2-dicarboxylic acid hydrazide using straight chain alkyl groups as spacers between the label and the ligand.
While these chemiluminescent compounds have been shown to be suitable labels, they are hydrophobic and, thereby, increase the hydrophobicity of the ligand complex. For example, column chromatographic purification of synthesized thyroxin-naphthalene-1,2-dicarboxylic acid hydrazide conjugates requires the use of organic solvents (U.S. Pat. No. 4,331,808). A documented disadvantage of an increased hydrophobicity is an increase in nonspecific binding effects. This results in a decreased signal to background ratio of immunoassays.
U.S. Pat. No. 4,645,646 describes the use of hydrophilic chain-like polymers with recurring functional groups (e.g., proteins) as carriers of multiple chemiluminescent luminol molecules to improve the sensitivity of luminescence immunoassays. However, these conjugates exhibit non-specific binding properties. In fact, some of the disclosed conjugates exhibit such marked non-specific binding properties to preclude their use in immunoassays (U.S. Pat. No. 4,645,646). These examples demonstrate that currently available techniques for coupling chemiluminescent labels to ligands pose an inherent problem. The hydrophobic properties of the chemiluminescent labels decrease the water solubility of the ligands to an extent that the sensitivity of the immunoassay is significantly reduced due to high non-specific background binding.
Labeling of ligands with fluorescent molecules poses very similar problems. Evrain et al., Steroids, vol. 35, 611-619 (1980) describe the synthesis of three fluorescein-labeled derivatives of testosterone using either cysteamine, or 1,3-diaminopropane, or 1,7-diaminoheptane as spacer between the fluorophore and the ligand. All derivatives proved to be highly hydrophobic. For example, analysis of the testosterone fluorescein conjugates by thin-layer chromatography on silica gel required the use of a combination of benzene:ethyl acetate:acetone (1:8:1) or chloroform:ethanol (7:3) as the solvent system. The hydrophobic nature of this compound is typical of fluorescein-labeled molecules and generally leads to high background readings in immunoassays.
U.S. Pat. No. 4,670,406 describes the use of bifunctional aromatic compounds (e.g., paranitrophenylisocyanate) as rigid coupling compounds for the synthesis of labeled ligands such as fluorescein-labeled digoxin. These rigid coupling reagents are advantageous in that they do not permit the fluorescent marker to "fold back" onto the ligand, thereby minimizing the possibility of quenching of the fluorescent compound by the ligand. However, as a result of their hydrophobic properties, the rigid coupling reagents further contribute to the loss of the water-solubility of the ligands upon labeling with fluorophores.
U.S. Pat. No. 4,452,886 describes the synthesis of ligand-containing polymers as carriers of multiple photon emitting (fluorescent) compounds. The disclosed polymers include proteins and synthetic or natural polypeptides having a large number of diamino acids for covalent attachment of photon emitting compounds or polymers of such compounds. Similar approaches have also been described in other publications. U.S. Pat. No. 4,604,364 discloses tracer compositions for immunoassays which contain photon emitting compounds coupled to ligands via an intermediate support material such as a protein or polypeptide. U.S. Pat. Nos. 4,166,105 and 4,169,137 describe antigen detecting reagents which are prepared by covalently linking fluorescent dye molecules to an appropriate antibody through a polymeric backbone having reactive functional groups along the length of its chain. Polymer backbone molecules reported to be suitable are polyethyleneimines (molecular weight range 1200 to 60,000 daltons), polypeptides such as polylysines, polyamides such as nylon-6, and low molecular weight (100 to 10,000 daltons) polymeric carboxylic acids. While such polymers help to reduce the loss of antibody binding activity upon fluorescent labeling, they are not suitable to compensate sufficiently for the hydrophobic properties of fluorescent labels such as fluorescein. As a result, such antigen detecting reagents exhibit significant non-specific background binding, thereby limiting the signal to background ratio of immunoassays. This is clearly demonstrated by a recent observation, in which antibody molecules labeled with only three fluorescein residues per antibody exhibited an approximately ten-fold higher non-specific binding than the same antibodies labeled with iodine-125 (personal communication, Lisa Shriver-Lake (1990)).
______________________________________ Specific and Non-specific Binding for DTAF- and .sup.125 I- Labeled Goat IgG (Shriver-Lake (1990)). Amount of Amount of Labeled Labeled Antigen Bound Antigen Bound to Immobilized to Immobilized Ratio of anti-Goat IgG Non-immune IgG Specific to Labeled (Specific (Nonspecific Nonspecific Antigen Binding) Binding) Binding ______________________________________ .sup.125 I-labeled 447.9 ng 30.4 ng 14.73 Goat IgG DTAF-label- 12.97 FU 9.52 FU 1.36 ed Goat IgG (3.1 DTAF/ IgG) ______________________________________ FU: arbitrary fluorescence units; DTAF: 5(4,6-dichlorotriazinyl)aminofluorescein. The specific and nonspecific binding ratios for the DTAFlabeled goat IgG and the .sup.125 Ilabeled goa IgG were measured using affinity purified antigoat IgG (Jackson ImmunoResearch, West Grove, PA) covalently attached to cover slips by Nmaleimidobutyryloxy succinimide ester (GMBS) as described in Bhatia et al, Anal. Biochem., vol. 178, pp. 408-413 (1989).
Thus, even a highly hydrophilic polypeptide chain such as an antibody with a molecular weight of approximately 150,000 apparently cannot compensate for the hydrophobic properties of as few as three fluorophore molecules.
Accordingly, there remains a need for molecular carriers which can link fluorescent or chemiluminescent labels to ligands, such as antigens and haptens, or receptors, such as antibodies, specific binding proteins and cell surface receptors, which do not suffer from the above-mentioned drawbacks. In particular, there remains a need for fluorescent-labeled and chemiluminescent-labeled conjugates which possess good water solubility, and for compounds which contain a plurality of fluorescent or chemiluminescent labels or sites for attaching such labels and which may be conveniently covalently bonded to a ligand, such as an antigen, hapten, or a receptor, such as an antibody, etc., to provide a ligand-label conjugate with good water solubility.